Inulin supplementation exhibits increased muscle mass via gut-muscle axis in children with obesity: double evidence from clinical and in vitro studies

Gut microbiota manipulation may reverse metabolic abnormalities in obesity. Our previous studies demonstrated that inulin supplementation significantly promoted Bifidobacterium and fat-free mass in obese children. We aimed to study gut-muscle axis from inulin supplementation in these children. In clinical phase, the plasma samples from 46 participants aged 7–15 years, were analyzed for muscle biomarkers before and after 6-month inulin supplementation. In parallel, the plausible mechanism of muscle production via gut-muscle axis was examined using macrophage cell line. Bifidobacterium was cultured in semi-refined medium with inulin used in the clinical phase. Cell-free supernatant was collected and used in lipopolysaccharide (LPS)-induced macrophage cell line to determine inflammatory and anti-inflammatory gene expression. In clinical phase, IL-15 and creatinine/cystatin C ratio significantly increased from baseline to the 6th month. In vitro study showed that metabolites derived from Bifidobacterium capable of utilizing inulin contained the abundance of SCFAs. In the presence of LPS, treatment from Bifidobacterium + inulin downregulated TNF-α, IL-6, IL-1β, and iNOS, but upregulated FIZZ-1 and TGF-β expression. Inulin supplementation promoted the muscle biomarkers in agreement with fat-free mass gain, elucidating by Bifidobacterium metabolites derived from inulin digestion showed in vitro anti-inflammatory activity and decreased systemic pro-inflammation, thus promoting muscle production via gut-muscle axis response. Clinical Trial Registry number: NCT03968003.


Subjects
This present study used the plasma and serum samples from the previously published randomized double-blinded placebo-controlled trial which conducted from August 2017 to July 2020 at the King Chulalongkorn Memorial Hospital (KCMH), Thailand 13 .The Institutional Review Board of the Faculty of Medicine, Chulalongkorn University approved the study protocol (IRB No. 240/60).All experiments were performed in accordance with relevant guidelines and regulations.Informed consent was obtained from all subjects and their legal guardians prior to study enrollment.This trial was registered at clinicaltrials.gov(NCT03968003).Thai children aged 7 to 15 years with obesity as a BMI more than 2 standard deviations (SDs) above median as per the WHO growth reference 16 were recruited as mentioned in the previous study 13 .

Study design
The detailed study design has been published elsewhere 13 .In brief, Thai children aged 7-15 years with obesity were randomly assigned to inulin extracted from Jerusalem artichoke by our patented technique (intervention), maltodextrin (placebo), and dietary fiber advice groups.All participants received monthly follow-up with the same standard advice of dietary intake and exercise for 6 months.The details and flow diagram of the study are reported elsewhere 13 .

The in vivo biomarkers of muscle building
The stored plasma and serum samples from the randomised controlled trial (RCT) of inulin supplementation in obese children were analyzed for biomarkers of muscle building at the 1st and 6th visits.Plasma and serum samples were stored at − 80 °C until analysis.For the present study, 56 plasma/serum samples (n = 46 from the inulin group, n = 5 from each placebo and dietary fiber advice group) were analyzed for myokine and creatinine/ cystatin C ratio to support the results of muscle building from the previous study 13 .Myokine, interleukin-15 (IL-15), was analyzed by enzyme-linked immunosorbent assay (ELISA).The quantification of IL-15, using the respective Human IL-15 ELISA kit (ab218266, Abcam, UK), was conducted in accordance with the prescribed manufacturer's protocols.Briefly, 50 µL of the sample was introduced into each well, followed by the addition of 50 µL of antibody, and incubation at room temperature for one hour.Subsequently, the sample mixture underwent three wash cycles using 350 µL of 1X wash buffer per cycle prior to the introduction of 100 µL of TMB development solution.The reaction proceeded at room temperature for 10 min and was subsequently terminated by the addition of 100 µL of stop solution.Measurement of the absorbance was carried out at a wavelength of 450 nm using a microplate reader.Furthermore, the concentration of creatinine in serum was measured using an enzymatic method on the Alinity C analyzers (Abbott Laboratories, USA), and that of cystatin C, N Latex Cystatin C, is an in vitro diagnostics kit containing reagents for the quantitative determination of cystatin C in human serum by means of particle-enhanced immunonephelometry using the BN Systems (SIEMENS, USA) with blood specimens stored at − 80 °C from the baseline and final visit.The samples were automatically diluted

The in vitro experiments
In parallel, the plausible mechanism of muscle mass building in the obese participants via gut-muscle axis was examined for in vitro phase.To test an influence of the metabolite from Bifidobacterium with or without inulin against macrophages, the condition media from Bifidobacterium (Bifidobacterium condition media; Bifido) with or without inulin were tested in a macrophage cell line (RAW264.7).
In Bifido preparation, Bifidobacterium longum HFDGO02 was cultured in SM medium using either glucose or inulin as the carbon source.Briefly, SM medium, glucose, and inulin were separately autoclaved.The medium containing either 1% (W/V) glucose or inulin was prepared and used for bacterial culture at 37 °C under an anaerobic condition for 48 h 17 .The supernatants were then collected by centrifugation and filtered (0.22-µm membrane filter) (Minisart; Sartorius Stedim Biotech GmbH, Göttingen, Germany), and 500 µl of the preparation was concentrated by speed vacuum drying at 40 °C for 3 h (Savant Instruments, Farmingdale, NY).The cell-free concentrated pellets were resuspended in an equal volume of DMEM or further diluted by DMEM into 50 and 25% dilution of the preparation and stored at − 20 °C until use.Then, murine macrophages (RAW264.7;ATCC-TIB-71) (Manassas, VA, USA) were cultured Dulbecco's modified Eagle medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum and 1% Penicillin-Streptomycin at 37 °C under 5% CO 2 before use.After that, the macrophages at 1 × 10 5 cells/well in 6-well plates were incubated for 24 h with several conditions (DMEM, inulin, Bifido, or Bifido with inulin) with or without LPS, using the LPS from Escherichia coli 026:B6 (Sigma-Aldrich, St. Louis, MO, USA) at 100 ng/ml.Notably, the dose of LPS to activate macrophages used here was followed our previous protocol [18][19][20] .After that, the expressions of several markers were measured by real time-qualitative polymerase chain reaction (RT-qPCR) using the primer presented in Table 1 with an established protocol.The RNA was extracted from the cells with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) together with RNeasy Mini Kit (Qiagen, Hilden, Germany) as 1 mg of total RNA was used for cDNA synthesis with iScriptreverse transcription supermix (Bio-Rad, Hercules, CA, USA).Quantitative real-time PCR was performed on a QuantStudio 5 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA).Expression values were normalized to Beta-actin (β-actin) as an endogenous housekeeping gene and the fold change was calculated by the ∆∆Ct method.Additionally, supernatant cytokines of macrophage pro-inflammatory activity, including tumor necrosis factor-α (TNF-α), IL-6, and IL-1β, alone with anti-inflammatory function, including transforming growth factor-β (TGF-β) and IL-10, were also evaluated by ELISA assay followed the manufacturer's protocols (R&D Systems, Inc., Minneapolis, MN, USA).

Statistical analysis
Wilcoxon signed-rank test was used to evaluate the difference in the change of variable outcomes between baseline and the 6th month, which included muscle biomarkers, IL-15 and creatinine cystatin C ratio, in clinical Table 1.Lists of primers used in the study.www.nature.com/scientificreports/phase.The gene expressions of inflammatory and anti-inflammatory markers in macrophage cell lines, presenting with or without LPS, among the Bifidobacterium and inulin controls, and treated with Bifidobacterium + inulin were evaluated using One-way ANOVA with Tukey analysis for in vitro phase.The alpha level of 0.05 was considered statistically significant for all analyses.All analyses were conducted using SPSS version 28.0 (SPSS Inc., Armonk, NY, USA) and GraphPad Prism version 9.0 (GraphPad Software, Boston, MA, USA).

The change in biomarkers of muscle building in clinical study
The stored plasma and serum samples from the children with obesity who received inulin supplementation (mean age: 10.3 ± 2.1 years, 54% male), placebo, and dietary fiber advice were analyzed.As mentioned above, in our previously published RCT of supplementation with inulin on body composition, we found that inulin supplementation increased fat-free mass in children with obesity 13 .IL-15, a myokine linked to muscle building, significantly increased in the inulin group (p < 0.0001, 95% CI 10.9-18.6).Moreover, within group analysis showed that (p = 0.0066, 95% CI 0.005-0.05)(Fig. 1a,b).The changes of IL-15 and creatinine/cytatin C ratio in the placebo and dietary fiber advice groups were not observed.

The change in pro-inflammatory and anti-inflammatory macrophage responses in cell line study
Our in vitro phase demonstrated that metabolites derived from Bifidobacterium capable of utilizing inulin contained the abundance of short chain fatty acids (SCFAs) (Supplement Fig. 1).In the presence of LPS, treatment of the cell-free supernatant from Bifidobacterium + inulin significantly downregulated pro-inflammatory genes, including TNF-α and IL-6 expression compared to the media control, Bifidobacterium alone, inulin alone, and the LPS (p < 0.05) (Fig. 2a,b).Downregulation of IL-1β and inducible nitric oxide synthase (iNOS) after Bifidobacterium + inulin administration was also significantly different from the LPS (p < 0.05) (Fig. 2c,d).Although inulin and Bifidobacterium supernatant alone reduced supernatant TNF-α and IL-6 compared to LPS control, Bifidobacterium + inulin further decreased both cytokines (p < 0.05) (Fig. 2e,f).On the other hand, only Bifidobacterium + inulin but not each factor in separation attenuated LPS-activated IL-1β in macrophages (p < 0.05) (Fig. 2g).For the anti-inflammatory responses, treatment from Bifidobacterium + inulin significantly upregulated FIZZ-1 and TGF-β expression compared to the LPS (p < 0.05) (Fig. 3a,b).No significant upregulation of,Arginase-1, and IL-10 expression (Fig. 3c,d) and alteration of supernatant anti-inflammatory cytokines (TGF-β and IL-10) (Fig. 3e,f) was observed after Bifidobacterium + inulin administration compared with LPS control.Dose effect of Bifidobacterium longum fermentative inulin (Bifido in Inulin or Bifidobacterium + inulin) for macrophage responses was performed using no dilution, 50% dilution, and 25% dilution by culture media (DMEM).Compared to LPS group, treatment with Bifidobacterium + inulin without dilution significantly downregulated gene expression of TNF-α, IL-6, IL-1β, and iNOS and reduced production of supernatant cytokines (TNF-α, IL-6, and IL-1β), while the dilution attenuated only IL-6 expression and supernatant IL-6 (Fig. 4a-g).The signal pathway diagram of the relationship between inflammation and microbiota-muscle axis was illustrated in Supplement Fig. 2.

The change in cell energy status in cell line study
Because of a possible relationship between cell energy status and macrophage functions, mitochondrial and glycolysis activities were determined through oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively (Fig. 5a,b).For mitochondrial activities, the OCR of macrophages was decreased by all activations; however, LPS and LPS plus inulin demonstrated the most severe reduction of mitochondrial functions (maximal respiration) (Fig. 5c).The mitochondrial activities were improved with the presence of www.nature.com/scientificreports/Bifidobacterium supernatant regardless of inulin (Fig. 5c), implying some beneficial molecules from the probiotics.For glycolysis status, only LPS and LPS plus inulin elevated glycolysis (Fig. 5d) which might be correlated with LPS-induced pro-inflammatory macrophage activity (Fig. 2).Nevertheless, the LPS-enhanced maximal glycolysis was reduced into the control regular state with incubation by supernatant of Bifidobacterium, alone or with inulin (Fig. 5d).In comparison with the control, the LPS-induced glycolytic stress effect in Bifidobacterium and Bifidobacterium + inulin supernatants was not observed (Fig. 5d), suggesting the influence of some bifidobacterial molecules on glycolysis.

Discussion
Obesity is a worldwide health problem and links with dysbiosis of the gut microbiota 1,7 .Pediatric obesity is associated with compromised muscular capacity and fitness 21 .Sarcopenic obesity is an arising issue in this population characterized by a significant decline in muscle mass alongside excessive fat accumulation 22 .We studied the benefits of the inulin extracted from Thai Jerusalem artichoke on children with obesity and found a significant increase in fat-free mass after a 6-month period of inulin supplementation 13 .In the present study, we examined the mechanism underlying gut-muscle axis from inulin supplementation on muscle mass in children with obesity.The in vivo study found a significant increase in IL-15 and creatinine/cystatin C ratio in the inulin supplementation group.For the in vitro study of inflammatory and anti-inflammatory gene expression, a significant downregulation of TNF, IL-6, IL-1β, and iNOS expression in the treatment with Bifidobacterium plus inulin was observed, whereas a significant upregulation of FIZZ-1 and TGF-β as the anti-inflammatory gene expressions was found.Furthermore, there were no significant differences in cellular glycolytic capacity between the treatment with Bifidobacterium plus inulin and controls.www.nature.com/scientificreports/Several studies in adults showed that supplementation with prebiotics, probiotics, and synbiotics improved muscle mass and strength [23][24][25][26][27] .Some studies were conducted on animal models to examine muscle mass and strength, but they did not specifically explore muscular biomarkers 28,29 .As far as we are aware, there has not been research done in children regarding the effect of prebiotics on muscular biomarkers via gut-muscle axis; therefore, our study might be the first to support this hypothesis.IL-15 was proposed as a contraction-induced myokine acting on local skeletal muscle to improve energy metabolism 30 .O'Leary et al. found that IL-15 played a role in myogenesis and protected muscles from degradation in isolated human myogenic cultures 31 .Another study revealed that exercise leads to higher serum IL-15, contributing to improved physical fitness 32 .In addition, IL-15 has been investigated for its role in indicating cancer-induced cachexia and its potential to prevent sepsis-induced muscle wasting 33,34 .Serum creatinine/cystatin C ratio was also offered as a biomarker of skeletal muscle status 35 .Basically, creatinine and cystatin C are used for estimating glomerular filtration.In contrast to creatinine, which is solely a product of muscle catabolism, cystatin C is produced from all nucleated cells and remains unaffected by muscle mass.Kashani et al. firstly investigated the benefits of serum creatinine to cystatin C ratio, so called the sarcopenia index, for assessing muscle mass 36 .Studies have highlighted the potential of this ratio in predicting severity, muscle mass, muscle strength, and muscle-adjusted visceral fat mass in non-alcoholic fatty liver disease patients 37,38 .In the case of the elderly, the researchers found the usefulness of creatinine/cystatin C ratio in assessing sarcopenia in terms of muscle mass and strength 39 .Therefore, based on the previous literatures, all the proxies of myogenesis from our study indicated inulin-induced muscle mass production and corresponded with increased fat-free mass in the children with obesity.
Systemic inflammation contributes to organ damage, including skeletal muscle loss, thereby increasing the risk of sarcopenia in children with obesity 22 .The study by Kalinkovich et al. revealed that a muscle mass reduction was caused by adipose tissue inflammation along with decreased uptake of fatty acids which led to an increased deposition of lipids within skeletal muscle tissue.This, in turn, could result in a decline in skeletal muscle mass 40 .Another previous study illustrated that change in the composition of gut microbiota, resulting in heightened absorption of bacterial products like LPS, could induce chronic inflammation and muscle production through elevated levels of inflammatory cytokines such as IL-6 and TNF-α, and then TNF-α triggers the activation of NF-κB and muscle ring finger-1, which in turn promotes increased ubiquitination of muscle proteins.This cascade subsequently leads to the breakdown of actin and myosin myofilaments, culminating in a reduction of muscle mass 41 .Additionally, there are other potential pathways, such as the production of mediators by the gut microbiota, including SCFAs, which play a role in positively influencing skeletal muscle production.The administration of butyrate, recognized for its anti-inflammatory and muscle-building properties attributed to its ability to inhibit the enzyme histone deacetylase, facilitates improved muscle building 42 .Our in vitro study showed that metabolites produced by Bifidobacterium, which can utilize inulin, were rich in SCFAs, and for in vitro study in macrophage cell lines, treatment with Bifidobacterium together with inulin significantly downregulated the key inflammatory gene expression, TNF-α and IL-6, and significantly decreased the expression of M1 macrophage markers, IL-1β and iNOS.These findings align with a study by Nicolucci et al. which reported a significant reduction in IL-6 with inulin supplementation compared to the control group.However, contrary to our results, they found no significant difference in lean mass gain in the inulin group 10 .Kelishadi et al. found a significant reduction of TNF-α and IL-6 in the synbiotic supplementation group 43 .The finding corresponded to our study reflecting the potential of rebalancing the gut microbiota dysbiosis leading to decreased inflammation.Interestingly, Bifidobacterium in DMEM (the glucose containing culture media) demonstrated a less potent anti-inflammation than Bifidobacterium with inulin (Fig. 2e-g) suggesting an impact of the different metabolites secreted from the probiotics after an incubation by glucose and inulin.Indeed, inulin stimulates the growth and activity of lactic acid bacteria as a bacterial nutrient source resulting in production of SCFAs, a beneficial factor for enterocyte to strengthen gut permeability 44 .More mechanistic studies are interesting.
Although we found Bifidobacterium could be an important link to promote muscle mass gain via gut-muscle axis, this is not limited only to this bacterium.Previous study reported that Bacteroides was positively associated with parameter of muscle function 41 , while a decrease in the genus Akkermansia was found in the gut microbiota of sarcopenia patients with cirrhosis 45 .
Dysbiosis of the intestinal microbiota impairs the gut barrier, enabling toxic substances derived from pathogenic bacteria, such as LPS, to enter the bloodstream.Activation of TLR4 by LPS can increase the levels of NF-κB and proinflammatory cytokines, including IL-6 and TNF-α 46,47 .Additionally, an initiation of inflammatory cascade in obesity via M1 macrophage that contributes to increased M1 macrophage markers, IL-1β and iNOS, leading to increased systemic inflammation and then affects muscle mass production 15,48 .Without LPS, Bifidobacterium condition media (with or without inulin) mildly upregulated expression of IL-1β and iNOS indicated some degree of inflammatory responses, perhaps due to the pathogen-associated molecular patterns (PAMPs) of bacteria.On the other hand, with LPS, the Bifidobacterium condition media downregulated these genes supporting the possible secretion of anti-inflammatory molecules.In the future, the separation of the antiinflammatory fraction from Bifidobacterium culture media might be interesting.However, inulin did not show inflammatory response because Bifidobacterium combined with inulin without LPS was not different from the Bifidobacterium culture media alone.Moreover, inulin itself might have an anti-inflammatory response because inulin alone with LPS downregulated IL-1β and iNOS (the genes of M1-pro-inflammatory polarization) 49 and upregulated FIZZ-1 and TGF-β, the anti-inflammatory M2 macrophage polarization genes 50 .Perhaps, inulin ameliorates LPS-TLR4 on macrophages by inhibiting their adhesion, leading to reduced inflammation 49 and promotes M2 macrophage polarization.In addition, SCFAs, induced by inulin might attenuate inflammation through increased mitochondrial function and muscle building 51 as indicated by increased fat-free mass and muscle biomarkers in our previous RCT 13 and this present study.Moreover, LPS reduced mitochondrial function and elevated glycolysis (extracellular flux analysis) supported the association between energy status and macrophage polarization 52 which was normalized by Bifidobacterium condition media.Because the cell energy status using Bifidobacterium with or without inulin was not different, inulin did not have an additional impact on cell energy status that was already improved by Bifidobacterium.Probiotic-induced anti-inflammatory macrophages might partly attenuate obesity-induced systemic inflammation through the strengthening of gut permeability [18][19][20] .
Indeed, there are several evidences of probiotic-induced anti-inflammation.For instance, Lactobacillus acidophilus was shown to counter inflammation through TGF-β1 signalling after Salmonella infection 53 .Fujii et al. investigated the anti-inflammatory properties of Bifidobacterium breve supplementation in preterm infants, which resulted in an observable augmentation in serum TGF-β1 levels 54 .Taken together, these might support our results of the anti-inflammatory role of prebiotics and probiotics, as we observed a trend towards elevated the gene expression of anti-inflammatory biomarkers, FIZZ-1 and TGF-β, in the current study.Furthermore, prebiotics like inulin might stimulate anti-inflammatory responses through alternative pathways.The incorporation of additional anti-inflammatory markers, such as peroxisome proliferator-activated receptor gamma, could yield supplementary evidence and further strengthen the findings 55 .To the best of our knowledge, this is the first study regarding the gut-muscle axis in children with obesity.The research was meticulously designed to elucidate the mechanisms underlying prebiotic-induced muscle building.All the results in myogenesis were the novel findings which were not found in the previous studies of prebiotics in obese children.Another strength is the method used in the study.We performed a clinical study according to our previous RCT study and the study was done in human subjects enhancing the potential for future practical applications.
In conclusion, the supplementation of inulin significantly promoted the biomarkers of muscle building in agreement with fat-free mass gain in children with obesity.This could be explained by Bifidobacterium metabolites derived from inulin digestion exhibited in vitro anti-inflammatory activity.Those metabolites may decrease systemic pro-inflammation, thus promoting muscle production in children with obesity via gut-muscle axis response.To bolster these results, future studies incorporating inulin alongside multi-strain probiotics may uncover further insights into the interaction between gut microbiota and muscle health in children with obesity.

Figure 5 .
Figure 5. Cell energy status.All activations reduced mitochondrial activities as indicated by maximal respiration (most prominence in LPS and LPS plus inulin), while only LPS and LPS plus inulin elevated glycolysis (p < 0.05).One-way ANOVA with Tukey analysis was used to evaluate parametric variables.Macrophages, RAW264.7 murine cell line; Oligomycin, an inhibitor against mitochondrial ATP synthesis; Antimycin A, a respiratory complex III inhibitor; Rotenone, a respiratory complex I inhibitor; 2-DG (2-Deoxyd-glucose), a glycolysis inhibitor.